Botulinum neurotoxins (BoNTs) are the most toxic substances known to humans, causing respiratory failure. Because of their lethality and ease of production, the Department of Defense (DoD) designates BoNTs as a category A bioterrorism agent. Developing effective, post-exposure antagonists to BoNT is among DoD's top priorities. Despite their lethality, BoNTs have cosmetic and pharmaceutical applications and are currently FDA-approved for treating glabellar lines (wrinkles), cervical dystonia, chronic migraines, blepharospasm, cranial nerve VII disorders, and cosmoses. BoNTs provide relief of muscle tension by silencing neurons that cause muscle contraction. For many disorders, BoNT-based treatments provide significant and long lasting pain reduction. BoNTs' exquisite specificity for neurons and long time of action make it a lead candidate for treating neurological and muscle disorders where other treatments have failed. Developing new BoNT-based therapies is hindered by the lack of assays to measure BoNT activity. Currently, the only widely accepted method involves injecting BoNT into mice and counting how many mice die after 1+ days. This process is slow, requires special facilities, and is very low-throughput; antagonist screening or developing new BoNT-derived therapeutics is prohibitively expensive and time-consuming. BioSentinel recently developed engineered cell lines that stably express a reporter for the detection of BoNT/A in living cells. These cell lines will become BoCell A, a cell-based assay for the detection of BoNT/A. The overall objectives of this Phase II proposal are to commercialize and validate the BoCell A assay and develop cell-based assays for detecting BoNT/B and E. Because BoNT has three activities-cell receptor binding and uptake, vesicle translocation, and target cleavage-a cell-based assay is required to effectively measure BoNT activity. BioSentinel developed a cell-based assay that measures BoNT activity by using fluorescence emission to monitor BoNT cleavage of SNARE proteins, the target of BoNT. BioSentinel will validate the BoCell A assay for biological relevance and high-throughput applications, and begin working to develop a second-generation BoCell A assay with sensitivity that matches that of the mouse bioassay. In addition, BioSentinel will build upon its Phase I accomplishment of identifying reporters that detect BoNT/B in living cells and engineer cell lines that stably express the reporters. Finally, the BoCell A assay will be engineered for the uptake and detection of BoNT/E. BioSentinel proposes to develop, validate, and commercialize assays that will enable researchers, clinicians, and pharmaceutical companies to rapidly quantify BoNT preparations, perform quality control measures, screen for new BoNT-based therapies, and screen for BoNT antagonists. Because of increased pressure for animal-free, high-throughput assays, a cell-based BoNT detection assay is greatly desired by researchers and industry, and would open a gateway to developing new BoNT-based therapies.